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1. Sequencing

As a starting point we strongly suggest that users read our Sanger Sequencing Guide. We also recommend that users follow our Sanger sequencing reaction protocol.

Successful Sanger sequencing is achieved by good template-primer complementary and template-primer ratio. The table below shows the recommended quantity of template to use in a sequencing reaction base on the size of the targeted template (extracted from the DNA Sequencing by Capillary Electrophoresis by Applied Biosystems).

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TEMPLATERECOMMENDED QUANTITY
PCR - 100 - 200bp
200 - 500bp
500 - 1000bp
1000 - 2000bp
>2000bp
1 - 3 ng
3 - 10ng
5 - 20ng
10 - 40ng
40 - 100ng
Single-Stranded DNA50 - 100ng*
Double-Stranded DNA (Plasmid DNA)200 - 500ng*
Large DNA (e.g. BACs,PACs,YACs,cosmids and fosmids)0.5 - 1.0ug
Bacterial genomic DNA2 - 3ug

NOTE: The AB3730 DNA Analyzer is a highly sensitive instrument, you may not need as much template as listed in the table above. Over-saturation of signal will cause a reduction id data quality.

1.2 Fragment Analysis

The most common issue with fragment analysis is that the size standard does not run properly (no size data) or under/over-saturation of the fluorescence signals. Good sample preparation for fragment analysis requires a fine balance between the quantity of the sample and the size standard, and if you are multiplexing, a fine balance between all the fluorescence labels and the size standard. Through proper optimisation, the data will have clean, beautiful peaks for subsequent analysis.

Excess reagents and salt can lower the quality of the data by interfering with the electrokinetic injection and the electrophoresis. Other contaminants, such as residual protein or detergents carried over from DNA extraction can adhere to the capillaries, thereby adversely reducing the quality of the data. Dilution (e.g. 1:100) or a clean-up step (e.g. ethanol precipitation) should be performed on the samples to minimise potential interferences of excess reagents, salt and other contaminants.

Over-saturated signals are difficult to analyse and have a similar negative impact as contaminants, therefore we strongly suggest our users to optimise their protocol prior to submitting large numbers of samples. 

2. Sequencing Guides

Below are links to resources for general information on Sanger sequencing and troubleshooting your data:

Ramaciotti Centre Sanger Sequencing Guide

DNA Sequencing by Capillary Electrophoresis by Applied Biosystems

Qiagen Guide to Template Purification and DNA Sequencing

3. Sequencing Clinic

The Centre runs a sequencing clinic whereby users of our service can book one on one time with a Sanger sequencing expert. Please contact us for further information or to make an appointment.

4. Software for Analysis

We advise our customers to always check the chromatograms manually to ensure correct base calling and genotype calling prior to subsequently analysis.

Software for Viewing and Editing Chromatograms:
Below is a list of analysis programs.  This list is by no mean comprehensive, there are vast number of programs available and each will have their own specific advantage.

  • Applied Biosystems Sequencing Analysis 5.1.1 for Windows XP and SeqScape (Sequencing Analysis and alignment) from Applied Biosystems.
  • FinchTV software for viewing and editing chromatograms (Macintosh OS X, Windows, Linux). Free access.
  • Geneious is a comprehensive suite of tools for analysis a range of sequencing data. Trial and student discount available.
  • Genemapper 5.0 for Windows 7 is available for Microsatellite, AFLP and RFLP analysis from Applied Biosystems.
  • Sequence Scanner (Sequencing Analysis) and Peak Scanner (Fragment Analysis) Applied Biosystems for PC. Free access.

Note: The Centre is not responsible for providing software for analysis. An analysis service is available (conditions and fees apply). Please contact us for further information.

 

 

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